The nuclear factor κB (NF-κB) family of transcription factors in mammals consists of homo- and hetero-dimeric combinations of five related proteins (p50, p52, p65/RelA, c-Rel, and RelB) that have a marked influence on the expression of numerous genes involved in immunity and inflammation, as well as cellular stress responses, growth, and apoptosis. Diverse pathways activate NF-κB, and control of these pathways is increasingly viewed as an approach to chemotherapy in the many diseases that have an associated inflammatory component, including cancer, stroke, Alzheimer's disease and diabetes.1-10 Activation of NF-κB occurs through multiple pathways. The classical pathway is triggered by binding of pro-inflammatory cytokines (TNFα and IL-1) and of a number of pathogens to several different receptors in the TNF-receptor and Toll-like/IL-1 receptor superfamilies. This leads to recruitment to the plasma membrane and activation of the IκB-kinase complex (IKK) consisting of IKKα and IKKβ kinases, and the scaffold protein NEMO/IKKγ, as well as a number of IKK-associated proteins. The main NFκB that is activated in the classical pathway is the p50/p65 heterodimer that exists in the cytoplasm as a complex with inhibitory protein IκBα. Activation of IKK primarily through IKKβ results in phosphorylation of IκBα on Ser32 and Ser36, followed by polyubiquitination and degradation of IκBα by the 26S proteasome, allowing p50/p65 to translocate to the nucleus.
Release of p50/p65 from IκBα also can be achieved by IKK-independent pathways triggered by DNA damage or oxidative stress that result in phosphorylation of IκBα on Ser residues other than Ser32 or Ser36, again leading to proteosomal degradation of IκBα. This signaling pathway involves a number of kinases including the MAP kinase p38 and casein kinase 2. There is also an oxidative stress pathway that phosphorylates IκBα on Tyr residues, leading to release of p50/p65 without proteosomal degradation of IκBα. Superimposed on the complex activation of p50/p65 is additional downstream regulation of the DNA-binding properties of p50/p65 through phosphorylation, acetylation and peptidyl-prolyl isomerization. Mostly this occurs in p65 and provides multiple points for control of NF-κB activation in a cell-specific and environment-specific manner. A wide range of kinases can phosphorylate p50/p65, which appears essential for the transactivation potential of p50/p65. This includes phosphorylation at many different sites, especially in p65, which adds to the complex regulation of NF-κB.4,10 
There are also alternative pathways to activation of NF-κB that result in formation of homo- or hetero-dimers other than p50/p65. A major alternative pathway, which is independent of IKKβ and NEMO, involves the IKKα homo-dimer whose activation is triggered by cytokines (other than TNFα), ligands such as CD40, and by certain viruses. This pathway requires recruitment of NF-κB-inducing kinase (NIK) with subsequent phosphorylation and activation of the IKKα homodimer. Activated IKKα phosphorylates p100, which is subsequently ubiquitinated and processed by the proteosome to p52. p52 and RelB then form a heterodimer that translocates to the nucleus. As with p50/p65, the p52/RelB heterodimer is further regulated by phosphorylation.4 
A large number of compounds including natural products have been reported to inhibit activation of NF-κB at one or more sites in the complex pathways of activation.11 This includes resveratrol (3,4′,5-trihydroxystilbene, 1), a polyphenolic
phytochemical that is found in numerous foods and is especially abundant in red wine. It has been proposed that the anti-oxidant activity of resveratrol is responsible for the French Paradox;12-14 this relates to the low incidence of cardiovascular disease in a French population with high intake of saturated fat.15 Both trans and cis isomers of resveratrol occur as phytochemicals, and both possess biological activities. Most studies of the biological activities of resveratrol and of synthetic stilbene analogs of resveratrol have focused on trans isomers. Resveratrol has been studied extensively in the context of carcinogenesis as a chemoprevention agent. All three stages of carcinogenesis, i.e., initiation, promotion and progression, have been reported to be inhibited by resveratrol.16 Because resveratrol exhibits anti-oxidant activity, which is based upon its phenolic groups, much of the research on resveratrol and on polyphenolic analogs of resveratrol has focused on anti-oxidant properties.17-21 In addition, the multiple biological activities reported for resveratrol, which in addition to its cardio-protective and anti-carcinogenic activity also includes inhibition of platelet aggregation, modulation of lipoprotein metabolism, anti-inflammatory and vasorelaxing activities,17,22-24 are often ascribed to the anti-oxidant properties of resveratrol. However, the oral bioavailability of resveratrol is low due to rapid metabolism, and the amount of resveratrol in dietary sources such as red wine is low compared to other polyphenols. Consequently, the circulating levels of resveratrol are low suggesting that the direct anti-oxidant effects of resveratrol are unlikely to explain its biological activities.12 Therefore, there has been extensive interest in the ability of resveratrol and other plant polyphenols to affect signaling pathways, including NF-κB.25 Signaling through NF-κB has been shown to be involved in the ability of resveratrol to induce heme oxygenase-1,26 inhibit phorbol ester-induced expression of COX-2,27 inhibit TNFα-induced proliferation of smooth muscle cells,28 enhance the radiosensitivity of lung cancer cells,29 and inhibit nitric oxide and TNFα production by LPS-activated microglia.30 Alzheimer's Disease
Alzheimer's disease (AD), the most common cause of dementia in elderly populations, currently afflicts almost 5 million people in the U.S., and this number is estimated to increase to 15 million by 2050. Most AD is sporadic with multiple risk factors, while some 10-15% is familial. It is well accepted that excessive production or diminished clearance of the Aβ peptide derived from the amyloid precursor protein (APP) is an essential factor in the etiology of AD. This is supported by studies of genetic mutations in APP in experimental animal models of AD as well as from studies of the genetics of familial AD.
There are two major neuropathological signatures of AD: extraneuronal amyloid plaques and neurofibrillary tangles. The plaques primarily consist of Aβ aggregates while the tangles consist of hyperphosphorylated tau protein. The exact mechanism by which these aggregates cause neuronal cell death remains to be established. However, considerable recent evidence points towards a major role for oligomeric forms of Aβ which are neurotoxic and can diffuse. Soluble Aβ is found in CSF of AD patients and correlates better with severity of disease than does the quantity of plaques. There are other common features of AD including the presence of chronic inflammation. The inflammatory response in brain is directed by activated microglia and reactive astrocytes. In normal brain, microglia are not activated. Under these conditions, neither pro-inflammatory signals nor reactive oxygen/nitrogen species (ROS/RNS) are formed. However, when microglia become activated in response to various insults, there is up-regulation of a number of surface receptors that promote phagocytotic activity by microglia. In addition, pro-inflammatory signals are released including interleukin-1β (IL1β) and tumor necrosis factor-α (TNFα) as well as ROS/RNS, thus contributing to the oxidative stress associated with AD. Activated microglia also associate with amyloid plaques. Microglia isolated from AD brain can scavenge Aβ. The considerable literature on the role of microglia in AD suggests that activation of microglia may contribute initially to clearance of Aβ aggregates, but that the chronic activation of microglia observed in AD leads to the neuropathological changes in the AD brain. Activated microglia also contribute to hyperphosphorylation of tau with development of neurofibrillary tangles, as well as to recruitment of activated astrocytes into the Aβ plaques.
It is now recognized that Aβ can increase the inflammatory response by activation of microglia and that the inflammatory response can contribute to Aβ deposition. Consequently there has been interest in hindering microglial activation as an approach to breaking this pathological cycle. Since activation of microglial results in release of ROS/RNS, attention has focused on use of anti-oxidants such as vitamin E. There are conflicting reports of the effects of anti-oxidants on development of AD, some supporting a role for anti-oxidants and others not supporting a role. Activation of microglia increases the oxidative burden in affected brain regions. However, how significant this increase is in contributing to neurodegeneration is not known. The field of anti-oxidant treatment of AD will need further controlled trials to assess this question.
Another area that has produced conflicting reports is the use of anti-inflammatory drugs, especially non-steroidal anti-inflammatory drugs (NSAIDS), in treatment of AD. COX-2, the inducible form of cyclooxygenase found in neurons and other cells and the source of pro-inflammatory eicosenoids, is up-regulated in AD brains. Overexpression of human COX-2 in mice results in age-related cognitive decline as well as neuronal apoptosis and astrocyte activation. The epidemiology studies of use of COX inhibitors (i.e. NSAIDs) by AD patients suggest that NSAID therapy may be useful. However, controlled clinical trials have been disappointing. These conflicting results may reflect the fact that the epidemiology studies begin with normal subjects and then assess risk of developing disease and whether this risk correlates inversely with drug use, whereas the clinical trials begin with subjects who have AD and look for improvement upon treatment. Other studies suggest that only a limited group of NSAIDs are effective and that these NSAIDs influence multiple targets in addition to COX-2. Animal model studies suggest that the dosing level of NSAID that is clinically feasible may not be sufficient to produce a pharmacological dose at the sites of plaque formation in AD brains.
Another area of interest in AD drug development focuses on signaling pathways that regulate expression of pro-inflammatory genes. Aβ stimulation of microglia results in up-regulation of the expression of TNFα and IL1 that is at least partly NFκB-dependent. IL1 is known to affect the expression of over 90 genes including those for cytokines, cytokine receptors, tissue remodeling enzymes and adhesion molecules. The mechanism for IL1 action involves activation of an IL1 receptor-mediated signal transduction pathway which leads to activation of NFκB. Thus NFκB is involved both in up-regulation of IL1 and in expression of the multiple genes regulated by IL1. These observations make inhibition of NFκB an attractive target for control of IL1-responsive genes in brain inflammation.
Diabetes
In 1998, it was suggested that the innate immune system is activated in diabetes, leading to a chronic inflammatory state that contributes to the disease process. More recently, there has been considerable support not only for an inflammatory contribution to diabetes but also to diabetic complications. Specifically, pro-inflammatory cytokines play a major role in microvascular complications. Endogenous production of TNF-α in vascular tissue is accelerated in diabetes where it contributes to increased vascular permeability in diabetic neuropathy. Both TNF-α and IL-1 expression are increased in diabetic retina where chronic low-grade inflammation appears to contribute to retinopathy. Likewise, diabetic nephropathy is associated with expression of inflammation markers such as CRP, fibrinogen and IL-6, and with increased expression of adhesion molecules such as ICAM-1, which promote inflammation by increasing leukocyte adherence and infiltration. The responses to these pro-inflammatory cytokines are especially prominent in endothelial cells (EC). Moreover, the response of EC to these cytokines commonly involves signaling through transcription factor NF-κB.
Oxidative stress has consistently been shown in experimental models of diabetes. Multiple mechanisms are involved that produce oxidative stress in EC in response to hyperglycemia, including: 1) protein glycosylation leading to AGE that trigger ROS production upon binding to the AGE receptor (RAGE); 2) glucose auto-oxidation; 3) accelerated metabolism of glucose through the aldose reductase/polyol pathway which consumes NADPH; 4) uncoupling of oxidative phosphorylation and of endothelial NO synthase (eNOS); 5) activation of specific isoforms of PKC; 6) increased flux through the hexosamine pathway; and 7) exposure to angiotensin II. Activation of NF-κB is often observed in response to these stresses. For example, exposure of EC to AGE generates ROS through activation of NADPH oxidase which then activates NF-κB followed by up-regulation of NF-κB-dependent cytokines and adhesion molecules. Angiotensin II can augment this process through crosstalk with the AGE-RAGE system, again involving NF-κB. High glucose can induce EC apoptosis through a PI-3-kinase-regulated expression of COX-2; this was shown to involve ROS and the NF-κB-regulated expression of COX-2. There has been considerable interest in a role for poly(ADP)-ribose polymerase (PARP) in EC dysfunction. PARP directly interacts with both the p50 and p65 subunits of NF-κB, suggesting that the role of PARP activation in diabetic complications is, at least in part, due to its interaction with NF-κB. Glucose-induced activation of NF-κB in EC is prevented by inhibitors of PKC, suggesting that the role of PKC in triggering the expression of pro-inflammatory cytokines is through downstream activation of NF-κB. There has also been considerable interest in mitochondria-derived ROS (specifically superoxide) produced in response to hyperglycemia and the relationship between these ROS and enhanced flux through the polyol pathway and the hexosamine pathway, PKC activation, and intracellular generation of AGE, all of which can be prevented by inhibiting the formation of mitochondria-derived ROS. The activation of these biochemical pathways appears to be due to ROS-induced activation of PARP, which results in inactivation of glyceraldehyde-3-phosphate dehydrogenase and subsequent accumulation of glycolytic intermediates that promote these pathways. It is noteworthy that inhibiting the production of mitochondria-derived ROS also prevents the activation of NF-κB, which may be related to the activation status of PARP. Clearly, activation of NF-κB appears to be a general feature of EC that are stressed by factors related to diabetic complications, suggesting a central role for NF-κB in EC dysfunction, especially as the key regulator of pro-inflammatory cytokines, adhesion molecules and extracellular matrix components, all of which are major players in diabetic microvascular complications.
The signaling mechanisms involved in inflammation that contributes to diabetes are under investigation, and are described by Wellen et al. (Wellen et al., J. Clin. Invest., 115, 1111-1119). This research indicates that inflammatory signaling pathways can be activated by metabolic stress or extracellular signaling molecules, and that endoplasmic reticulum stress (ER stress) leads to the activation of inflammatory signaling pathways and thus contributes to insulin resistance. Ozcan et al., Science, 306, 457-461 (2004). For example, several serine/threonine kinases are activated by inflammatory or stressful stimuli that contribute to inhibition of insulin signaling, including c-Jun N-terminal kinase (JNK) and I-κB kinase (IKK). The three members of the JNK group of kinases (JNK-1, -2, and -3) belong to the MAPK family and regulate multiple activities, in part through their ability to control transcription by phosphorylating activator protein-1 (AP-1). Loss of JNK1 has been shown to prevent the development of insulin resistance and diabetes in both genetic and dietary models of obesity.
A model of the overlapping metabolic and inflammatory signaling and sensing pathways in adipocytes and macrophages that influence diabetes and inflammation is provided by FIG. 2. As shown in FIG. 2, signals from various mediators converge on the inflammatory signaling pathways, including the kinases JNK and IKK. These pathways lead to the production of additional inflammatory mediators such as NF-κB and AP-1 through transcriptional regulation as well as to the direct inhibition of insulin signaling. Opposing the inflammatory pathways are transcriptional factors from the PPAR and LXR families, which promote nutrient transport and metabolism and antagonize inflammatory activity.